The field of the invention is amplifying RNA.
RNA is a frequent starting material for genetic analysis, such as microarray-based diagnostics and sequencing, and a wide variety of methods have been devised to amplify RNA, generally by first copying the RNA to cDNA and then using PCR and/or repeated rounds of transcription to obtaine an amplified product. For example, Silver et al. (1992) U.S. Pat. No. 5,104,792; Liang et al. (1997) U.S. Pat. No. 5,599,672; and Shuber (1999) U.S. Pat. No. 5,882,856 describe methods for amplifying RNA. The present invention provides an improved method of amplifying RNA which is adaptable to total RNA input, low quantity input (100 pg or less mRNA) and linear or quantitative PCR amplification.
The invention provides methods and compositions for amplifying RNA sequences. In one aspect, the invention comprises the steps of:
(a) hybridizing to a target RNA a first primer comprising a 3xe2x80x2 target RNA hybridizing sequence and a first 5xe2x80x2 defined amplifiable sequence;
(b) extending the first primer with a reverse transcriptase to form a first cDNA strand;
(c) hybridizing to the first cDNA strand a second primer comprising a 3xe2x80x2 random EDNA hybridizing sequence and a second 5xe2x80x2 defined amplifiable sequence;
(d) extending the second primer with a DNA polymerase to form a second cDNA strand; and
(e) amplifying the second cDNA strand with a third primer comprising the first 5xe2x80x2 defined amplifiable sequence.
In one principal embodiment, step (b) yields a heteroduplex of the target RNA and the first cDNA and flier comprises the step of digesting the target RNA of the heteroduplex with a RNase sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step. In various applications, the 3xe2x80x2 target RNA hybridizing sequence may be random or nonrandom, such as complementary to a predetermined sequence (e.g. a coding region, a polyA junction, or a polyA tail), and the first and second 5xe2x80x2 defined amplifiable sequences may be the same or different. In particular embodiments wherein the first and second 5xe2x80x2 defined amplifiable sequences are different, the method further comprises the step of functionally depleting the first primer between steps (b) and (c); step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5xe2x80x2 defined amplifiable sequence; and/or the method further comprises step (D amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation. In particular applications, the method may be practiced in a single tube (homogeneous assay).
In another principal embodiment, the 3xe2x80x2 target RNA hybridizing sequence is random; the first and second 5xe2x80x2 defined amplifiable sequences are different; and step (b) comprises the step of functionally depleting the first primer to prevent it from hybridizing with the first cDNA strand in subsequent steps.
In aspects of both principal embodiments, interference by the first and/or second primers with the amplification step (e) may be reduced by adding the third and/or fourth primer of step (e) in functional excess of the first and/or second primer; and/or functionally depleting remaining first and second primers between steps (d) and (e).
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more and the term xe2x80x9corxe2x80x9d means and/or.
The first step of the disclosed methods comprises (a) hybridizing to a target RNA a first primer comprising a 3xe2x80x2 target RNA hybridizing sequence and a first 5xe2x80x2 defined amplifiable sequence. A wide variety of target RNAs may be employed, including total cellular RNA, amplified RNA, purified RNA species such as rRNA, tRNA or preferably, mRNA, etc. The fist primer comprises a 3xe2x80x2 sequence of length and sequence sufficient to hybridize with the target RNA. Depending on the application, this 3xe2x80x2 hybridizing sequence may be random, specific or a combination of random and specific sequences. For example, a primer population comprising random 3xe2x80x2 hybridizing sequences provides a xe2x80x9cuniversalxe2x80x9d primer set capable of targeting any RNA species. In other embodiments, primers comprising polyT 3xe2x80x2 hybridizing sequences may be used to target polyA tails of mRNA; primers comprising predetermined specific sequences may be used to target particular, predetermined RNA species comprising complementary sequences; and primers comprising a random region joined to a wobble nucleotide (A, C or G) joined to a polyT region may be used to target mRNA polyA junctions. The first primer also comprises a first 5xe2x80x2 defined amplifiable sequence, which may be any sequences which can be used in the subsequent specific amplification step and preferably comprises a PCRable tag. Suitable reaction conditions for effecting hybridization between the target RNA and first primer are known in the art, readily ascertained empirically, and/or described and/or exemplified herein.
The second step of the methods comprises (b) extending the first primer with a reverse transcriptase to form a first cDNA strand. Depending on the application, an RNAse activity may be present during this step, which can effect the degradation of the original RNA template subsequent to, or coincident with reverse transcription, allowing, for example, priming of the new cDNA strand with the same primer. In a preferred embodiment, the RNAse activity is provided by the reverse transcriptase, such as Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase (both available from Promega, Madison, Wis.), Rous Associated Virus 2 (RAV2) and Human Immunodeficiency Virus 1 (HIV1) reverse transcriptases (both available from Amersham Pharmacia), etc. In applications where the 3xe2x80x2 target RNA hybridizing sequence is random and the first and second 5xe2x80x2 defined amplifyable sequences are different, RNAse activity is preferably avoided so that second strand cDNA synthesis does not occur in the same reaction mixture. Exemplary suitable reverse transcriptases without RNase activity include MMLV-RT RNase H minus (e.g Promega Catalog #M5301 and #M3682), display THERMO-RT (Display Systems Biotech, Vista Calif.), Strat-Script RT (Stratagene, San Diego, Calif.), etc.
The third step of the methods comprises (c) hybridizing to the first cDNA strand a second primer comprising a 3xe2x80x2 random cDNA hybridizing sequence and a second 5xe2x80x2 defined amplifiable sequence. Note that depending on the application, the second primer may have the same 3xe2x80x2 hybridizing sequence and/or the same 5xe2x80x2 defined amplifiable sequences as does the first primer, or one or both sequences may differ; see examples, below. For example, where the first and second 5xe2x80x2 defined amplifiable sequences are different, the method may also comprise the step of functionally depleting the first primer between steps (b) and (c). Functional depletion reduces interference of the first primer with the second primer extension reaction and may be effected by any convenient means such as removal (e.g. size exclusion or affinity chromatography), inactivation (e.g. hydrolysis, conjugation, etc.), etc.
The fourth step of the methods comprises (d) extending the second primer with a DNA polymerase to form a second cDNA strand. Suitable DNA polymerases and reaction conditions are known in the art, readily ascertained empirically, and/or described and/or exemplified below.
The fifth step of the method comprises (e) amplifying the second cDNA strand with a third primer comprising the first 5xe2x80x2 defined amplifiable sequence. To reduce interference from the first primer, the third primer of step (e) may be added in functional excess of the first primer, and/or remaining first (and/or second primer, if present and distinct from the first) may be functionally depleted between steps (d) and (e). Depending on the particular application, amplification step (e) may employ additional primers and reactions. For example, where the first and second 5xe2x80x2 defined amplifiable sequences are different, step (e) may further comprise amplifying the second cDNA strand with a fourth primer comprising the second 5xe2x80x2 defined amplifiable sequence. In a more particular embodiment of this application, the method further comprises step (f) amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation.
Preferred applications of the method reduce handling steps, such as wash steps, inherent in prior art methods, preferably to only a single wash step, more preferably to no wash steps wherein the method is practiced continuously, preferably homogenously, and in a single tube (i.e. container or reaction vessel).
The third and fourth primers comprise sequences identical to those of the defined sequence portions of the first and second primers and may contain optional detectable labels at positions other than their 3xe2x80x2 termini. The labels may be directly detectable, as in the case of fluorescent or radio labels, or indirectly detectable, as in the case of biotin, nitrophenol, or related labels for which there are high affinity specific binding reagents which contain directly detectable labels and which are used in second binding reactions to measure the presence of the indirect labels.
In a preferred mode, the sequences of the sequence specific portions of the first primer and the second primer are identical. In this mode, the third and fourth primers that are optionally labeled are also identical, such that only two primers become necessary for all amplification and labeling steps. In a further preferred mode, the hybridization temperature of the first primer portion that hybridizes to the mRNA and the second primer portion that hybridizes to the cDNA are between 20xc2x0 C. and 45xc2x0 C., and the hybridization temperature of the sequence specific portions of the first and second primers are between 50xc2x0 C. and 80xc2x0 C.
In yet other modes of this invention, labeling does not occur during the amplification process, but is done after amplification. In this mode, the amplification products can be labeled by a variety of methods including the incubation of reactive label reagents with sites on the DNA strands that include the terminal hydroxyl group, exocyclic amines of the DNA bases, and the bridging internucleotide phosphate groups. Alternatively, labels may be incorporated by the process of nick-translation employing appropriately labeled nucleotide triphosphates and an appropriate DNA polymerase such as the Klenow fragment.
A wide variety of materials and methods are known in the art for arraying polynucleotides at discrete elements of substrates such as glass, silicon, plastics, nylon membranes, etc., including contact deposition, e.g. U.S. Pat. Nos. 5,807,522; 5,770,151, etc.; photolithography-based methods, e.g. U.S. Pat. Nos. 5,861,242; 5,858,659; 5,856,174; 5,856,101; 5,837,832, etc; flow path-based methods, e.g. U.S. Pat. No. 5,384,261; dip-pen nanolithography-based methods, e.g. Piner, et al., Science Jan. 29, 1999: 661-663, etc.; etc. In a preferred embodiment, the capture polynucleotides are arrayed at corresponding discrete elements in high density, generally at least 100, preferably at least 1000, more preferably at least 10,000, most preferably at least 100,000 discrete elements per square centimeter.
In one principle application of the method, step (b) yields a heteroduplex of the target RNA and the first cDNA and further comprises the step of digesting the target RNA of the heteroduplex with a RNase sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step. In particular embodiments of this application, the first and second 5xe2x80x2 defined amplifiable sequences are the same and the 3xe2x80x2 target RNA hybridizing sequence is random; the first and second 5xe2x80x2 defined amplifiable sequences are the same and the 3xe2x80x2 target RNA hybridizing sequence is nonrandom; the first and second 5xe2x80x2 defined amplifiable sequences are the same, the 3, target RNA hybridizing sequence is nonrandom and the method is practiced in a single tube; the 3xe2x80x2 target RNA hybridizing sequence is nonrandom, the first and second 5xe2x80x2 defined amplifiable sequences are different and the method further comprises the step of functionally depleting the first primer between steps (b) and (c); the 3xe2x80x2 target RNA hybridizing sequence is nonrandom, the first and second 5xe2x80x2 defined amplifiable sequences are different, the method further comprises the step of functionally depleting the first primer between steps (b) and (c) and the method is practiced in a single tube.
In a second principle application of the method, the 3xe2x80x2 target RNA hybridizing sequence is random; the first and second 5xe2x80x2 defined amplifiable sequences are different; and step (b) comprises the step of functionally depleting the first primer to prevent it from hybridizing with the first cDNA strand in subsequent steps. Exemplary protocols for representative examples of these principle applications are provided below.